This application is directed to oligonucleotides and oligonucleosides functionalized to include lipophilic moieties. Relative to their unfunctionalized parent compounds, such lipophilic oligonucleotide conjugates have improved biostability and altered biodistribution in mammals. In one embodiment, such lipophilic oligonucleotide conjugates are used in a method of targeting antisense oligonucleotides to hepatic tissues and thereby preferentially modulating gene expression in the liver and associated tissues of a mammal.
Messenger RNA (mRNA) directs protein synthesis. Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
The naturally occurring events that provide the disruption of the nucleic acid function, discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (Miller, et al., Anti-Cancer Drug Design, 1987, 2, 117) and xcex1-anomer oligonucleotides are examples of antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research reagents and potential therapeutic purposes. At least for therapeutic purposes, and for research purposes involving whole cells, tissues or animals, the antisense oligonucleotides and oligonucleotide analogs must be transported across cell membranes or otherwise taken up by cells in order to exhibit activity. One method for generally increasing membrane or cellular transport is by the attachment of a pendant lipophilic group. More specifically, Ramirez et al. (J. Am. Chem. Soc., 1982, 104, 5483) introduced the phospholipid group 5xe2x80x2-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3xe2x80x2 and 5xe2x80x2 positions. Subsequently Shea et al. (Nuc. Acids Res., 1990, 18, 3777) disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5xe2x80x2-phosphate on the 5xe2x80x2-terminus of the oligonucleotide. Certain of the Shea et. al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra et al. (Tetrahedron Letters, 1987, 28, 3581).
In other work, a cholesteryl group was attached to the inter-nucleotide linkage between the first and second nucleotides (from the 3xe2x80x2 terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further by Letsinger et al. (Proc. Natl. Acad. Sci. USA, 1989, 86, 6553). The aromatic intercalating agent anthraquinone was attached to the 2xe2x80x2 position of a sugar fragment of an oligonucleotide as reported by Yamana et al. (Bioconjugate Chem., 1990, 1, 319). The same researchers placed pyrene-1-methyl at the 2xe2x80x2 position of a sugar (Yamana et. al., Tetrahedron Lett., 1991, 32, 6347).
Lemairte et al. (Proc. Natl. Acad. Sci. USA, 1986, 84, 648) and Leonetti et al. (Bioconjugate Chem., 1990, 1, 149). The 3xe2x80x2 terminus of the oligonucleotides each include a 3xe2x80x2-terminal ribose sugar moiety. The poly(L-lysine) was linked to the oligonucleotide via periodate oxidation of this terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates are described in European Patent application 87109348.0. In this instance the lysine residue was coupled to a 5xe2x80x2 or 3xe2x80x2 phosphate of the 5xe2x80x2 or 3xe2x80x2 terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3xe2x80x2 terminus of an oligonucleotide to link a peptide to the oligonucleotide (Corey et al., Science, 1987, 238, 1401; Zuckermann, et al., J. Am. Chem. Soc., 1988, 110, 1614; and Corey et al., J. Am. Chem. Soc., 1989, 111, 8524).
Nelson et al. (Nuc. Acids Res., 1989, 17, 7187) describe a linking reagent for attaching biotin to the 3xe2x80x2-terminus of an oligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3xe2x80x2-Amine on. It is also commercially available under the name 3xe2x80x2-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy et al. (Tetrahedron Letters, 1991, 32, 879). A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5xe2x80x2-terminus of an oligonucleotide is 5xe2x80x2-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg et al. (Antisense Research and Development, 1991, 1, 161) to link fluorescein to the 5xe2x80x2-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3xe2x80x2-terminus of an oligonucleotide. Asseline et al. (Proc. Natl. Acad. Sci. USA, 1984, 81, 3297) described linking acridine on the 3xe2x80x2-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis et al. (Tetrahedron Letters, 1987, 28, 5199) report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3xe2x80x2 hydroxyl group of the 3xe2x80x2 terminal nucleotide of the oligonucleotide. Chollet (Nucleosides and Nucleotides, 1990, 9, 957) attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the 5xe2x80x2 terminal phosphate of an oligonucleotide. Chollet then used the bifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) to link an interleukin protein to the oligonucleotide.
An EDTA iron complex has been linked to the 5 position of a pyrimidine nucleoside as reported by Dreyer et al. (Proc. Natl. Acad. Sci. USA, 1985, 82, 968). Fluorescein has been linked to an oligonucleotide in the same manner as reported by Haralambidis, et al. (Nucleic Acid Research, 1987, 15, 4857) and biotin in the same manner as described in PCT application PCT/US/02198. Fluorescein, biotin and pyrene were also linked in the same manner as reported by Telser et al. (J. Am. Chem. Soc., 1989, 111, 6966). A commercial reagent, Amino-Modifier-dT, from Glen Research Corporation (Sterling, Va.) can be utilized to introduce pyrimidine nucleotides bearing similar linking groups into oligonucleotides.
Cholic acid linked to EDTA for use in radioscintigraphic imaging studies was reported by Betebenner et al. (Bioconjugate Chem., 1991, 2, 117); however, it is not known to link cholic acid to nucleosides, nucleotides or oligonucleotides.
Despite these efforts and other research in the field, it is not known in the art to use lipophilic conjugation to alter the pharmacodynamic properties of an antisense compound, i.e., an agent that works via a nucleotide sequence-dependent antisense mechanism.
It is one object of this invention to provide oligonucleotides and oligonucleosides functionalized to include lipophilic moieties in order to produce lipophilic oligonucleotide and oligonucleoside conjugates which, relative to their unfunctionalized parent compounds, have improved biostability and altered biodistribution in mammals.
It is a further object of the invention to provide methods of modulating gene expression in cells, tissue(s) or organ(s) of a mammal using the lipophilic oligonucleotide and oligonucleoside conjugates of the invention.
It is a particular object of the invention to provide compositions for and methods of targeting antisense oligonucleotides to hepatic tissues and thereby preferentially modulating gene expression in the liver and associated tissues of a mammal.
These and other objects are satisfied by the present invention, which provides oligonucleotides and oligonucleosides functionalized to include lipophilic moieties. In one aspect, the invention provides nucleosides having base portions and ribofuranosyl sugar portions. Such nucleosides bear at a 2xe2x80x2-O-position, a 3xe2x80x2-O-position, or a 5xe2x80x2-O-position a substituent having formula:
xe2x80x94RAxe2x80x94N(R1a)(R1b)
where:
RA is alkyl having from 1 to about 10 carbon atoms or RA is (CH2xe2x80x94CH2xe2x80x94Qxe2x80x94)x;
R1a and R1b, independently, are H, RA, R2, or an amine protecting group or have formula C(X)xe2x80x94R2, C(X)xe2x80x94RAxe2x80x94R2, C(X)xe2x80x94Qxe2x80x94RAxe2x80x94R2, C(X)xe2x80x94Qxe2x80x94R2;
R2 includes a steroid molecule, a reporter molecule, a lipophilic molecule, a reporter enzyme, a peptide, a protein, or has formula xe2x80x94Qxe2x80x94(CH2CH2xe2x80x94Qxe2x80x94)xxe2x80x94R3;
X is O or S;
each Q is, independently, is NH, O, or S;
x is 1 to about 200;
R3 is H, RA, C(O)OH, C(O)ORA, C(O)R4, RAxe2x80x94N3, RAxe2x80x94NH2, or RAxe2x80x94SH; and
R4 is Cl, Br, I, SO2R5 or has structure: 
m is 2 to 7; and
R5 is alkyl having 1 to about 10 carbon atoms.
In another aspect, the invention provides oligonucleotides and oligonucleosides comprising a plurality of linked nucleosides, wherein each nucleoside includes a ribofuranosyl sugar portion and a base portion and at least one (preferably more than one) of the nucleosides bears at a 2xe2x80x2-O-position, a 3xe2x80x2-O-position, or a 5xe2x80x2-O-position a substituent having formula xe2x80x94RAxe2x80x94N(R1a)(R1b).
In another aspect the invention provides methods for preparing oligonucleotides and oligonucleosides comprising the steps of contacting nucleosides according to the invention for a time and under reaction conditions effective to form a covalent bond therebetween. In preferred embodiments, at least one of the nucleosides bears a phosphoramidate group at its 2xe2x80x2-O-position or at its 3xe2x80x2-O-position.
In other embodiments, compounds according to the invention are prepared by contacting a nucleoside, oligonucleotide or oligonucleoside with derivatizing reagents. For example, a nucleoside, oligonucleotide or oligonucleoside bearing a 2xe2x80x2-hydroxy group, a 3xe2x80x2-hydroxy group, or a 5xe2x80x2-hydroxy group under basic conditions with a compound having formula L1xe2x80x94RAxe2x80x94N(R1a)(R1b) wherein L1 is a leaving group such as a halogen and at least one of R1a and R1b is an amine protecting group.
The present invention also provides methods for inhibiting the expression of particular genes in the cells of an organism, comprising administering to said organism a compound according to the invention. Also provided are methods for inhibiting transcription and/or replication of particular genes or for inducing degradation of particular regions of double stranded DNA in cells of an organism by administering to said organism a compound of the invention. Further provided are methods for killing cells or virus by contacting said cells or virus with a compound of the invention. The compound can be included in a composition that further includes an inert carrier for the compound.